In order to understand the molecular mechanism of the light-induced proton pump of the purple membrane (PM), both the photoreceptor protein bacteriorhodopsin (BR) and the membrane lipids were chemically modified. Reaction products were assayed for proton pump activity by studying light-induced steady-state changes in proton binding delta minus over delta (h). Chemical modification: Carboxyl groups were modified by reaction with ethyldimethylaminopropylcarbodiimide (EDC). Reaction with EDC at pH 8 appears to inhibit the proton pump. At pH 4.5, EDC causes crosslinking of BR, involving carboxyl groups in the C-terminal region (residues 23l-247). At pH 5.5, EDC couples a fluorescent probe, dansyl hydrazine (DH), to the C-terminal region and also to a second site. An amino group within the first 7l amino acids reacts specifically with dansyl chloride at pH 8. Tyrosine was nitrated by tetranitromethane at pH 8 in detergent solution, and the product displayed pump inhibition. Steady-state pH changes: A simple 3-state model was shown to account for variation of delta minus over delta h with pH, temperature, light intensity, wave-length, and optical density. The apparent activation energy for proton uptake was l5 kcal/mol, suggesting a process in addition to proton transfer is involved in this step.